An improvement in the method of purifying enzymes and peptides that is continuous, one step in operation, and modularized, which utilizes polyacrylamide gel as a medium for continuous separation but does not make use of a flowing of the buffer through the gel. The method is continuous, in that the flow of the stream of crude enzymes and peptides is not interrupted during any stage of the operation. The method is one-step in the operation of purification as the enzymes and peptides, from the time they are fed into the gel, undergo a process of purification which requires no additional steps of purification. The electrophoresis on the polyacrylamide gel results in high purification as the polyacrylamide is one unified piece of gel which purifies higher than any other substance because it provides a high molecular sieving resolution. The "Adjustable Specialized Geometrically Located Electrode System" results in purification of enzymes and peptides on a large scale, for the different geometrically shaped electrodes provide different pathways for each different enzyme and peptide existing in the crudely applied mixture thereby enabling the separation, elution, and collection of typical enzymes and peptides, though as many as one hundred different enzymes and peptides are caused to flow from the crude protein mixture through the gel at the same time. The modularization of the interchangeable parts of the electrode system, which include such geometrically different shapes as diagonal linear electrodes, arced electrodes, parabolic electrodes, point and ball electrodes, and other shapes, create a diversity of field gradients by causing many different particle vectors, and permit a multifunctional versatile implementation and application of electrophoresis in the purification of the enzymes and peptides. The automatic switching system of the electrodes, which last from 1/100th of a second to 10 seconds, and the on and off repetitive selectivity of the electrodes, enable the prevention of the electrode system's interference on one another, and enable the avoidance of short circuits, thereby providing the creation of required potential vectors. The "Adjustable, Specialized, Geometrically Located Electrode System" containing the innovative features of modularization of electrode systems, automatic switching, repetitive and selective operations of the divergent geometrically shaped electrode systems, and the divergent geometrically shaped electrode systems, have not been used in the past or present methods of electrophoresis.
The method allows a high purification simultaneously as it allows purification on a large scale, dual factors which do not exist together under present methods of electrophoresis. The method conceives a separation in a rectangular thin gel placed between the separate independent fields perpendicular to each other. The two electric streams are not operated simultaneously, but rather intermittently, during short periods of time, switched automatically by controlled automatic switches. However the gel itself is under a convertable single electrical field continuously. The intensity of one field or both fields, is in a gradient form alongside the width of the field applied onto the gel layer. This is accomplished by introducing electrodes, forming the respective fields, in such a way that the resistance of the medium is gradually changed along the width of the field. These changes are adjusted to follow many different curves such as linear, exponential, hyperbolic, circular and other shapes, by altering the geometric location and shapes of these electrodes, the theory being that a greater rate of change in the field yields a finer separation of particles. A typical configuration of the linear gradient can be achieved by placing electrodes diagonally alongside the gel and using the variations in diagonal to control the slope of the gradient. The exponential gradient may be achieved in the same manner as the linear but using an exponential curve in the electrodes. The point or ball electrodes in an exponential system consist of electrodes in small circles of buffers, one for positive and one for negative. These electrodes when placed as indicated (in FIG. 2) produce a field which has an exponential effect on the particle accelerations thereby yielding better particle separation. Other variations such as concave and convex hyperbolic curves have proven to yield beneficial results. The enzymatic preparation mixture is introduced into the gel continuously by a very slow constant flow, through a thin tubing into a hole crossing the thickness of the gel, during the application of the electrical fields. The vertical electrical field is the "separator" of proteins according to their mobility in the field on account of the net charge, molecular dimension, and molecular configuration, by applying upon them a vertical vector (A). The horizontal electrical field applies a vector (B) and causes the proteins to move horizontally. The proteins pass through the gel via a series of tiny holes across the thickness of the gel, eluted by the flowing buffer which runs during the operation. The gradient is the factor which enables the faster fractions to emerge sooner and closer to the application point than the slower ones. An ambient temperature is maintained at any degree needed by circulation of coolant around a vertical plate from a refrigerated bath, permitting preparative separations for heat labile substances such as enzymes. A combinational system is used to yield better separation. Each band that is separated is run through higher voltage, an electrode separation having better differentiation to obtain a finer yield. The advantage is that a smaller system is required for a greater batch of input. Another advantage is that less heat is dissipated by the system due to the lower voltage required for a smaller system.